Allograft rejection is dependent upon T cell activation. While required for activation, stimulation of the T cell through the T cell receptor (TCR) is not by itself sufficient, as a second signal is also needed. The best characterized second or costimulatory signal is mediated by the T cell surface molecule CD28 interacting with its ligands, B7-1 and B7-2, present on the surface of antigen presenting cells (APCs). CTLA4Ig, a soluble fusion protein which binds both B7-l and B7-2, acts as an inhibitor of CD28 signaling and in a cardiac model of allogeneic transplantation, the combination of donor-specific transfusion (DST) plus CTLA4Ig prevents allograft rejection. Interestingly, delaying CTLA4Ig administration until 2 days after transplantation results in indefinite graft survival, whereas CTLA4Ig injection at the time of transplantation does not. Thus, delaying T cell costimulatory blockade until after antigenic stimulation is essential for tolerance induction. This proposal seeks to examine the mechanism(s) by which DST plus delayed blockade of costimulation induces tolerance as a means to develop the skills necessary for an independent career in immunologic research. Under the sponsorship of Dr. Turka, an experienced investigator in the area of CD28-mediated immune responses, and with the resources available to us at the University of Pennsylvania, particularly the immunology faculty, Dr. Judge will investigate the roles of chimerism and CTLA4Ig-mediated immunosuppression in the establishment of allo-specific tolerance in this model. In Specific Aim #1, we will determine if DST-induced chimerism is required for tolerance. The ability of DST plus CTLA4Ig to induce chimerism will be examined by flow cytometry and PCR. The requirement for chimerism will be determined using DST cells which may be selectively ablated in vivo by using depleting antibodies or, in the case of DST cells expressing the herpes virus thymidine kinase gene, by infusion of ganciclovir. In Specific Aim #2, we will investigate potential mechanisms of tolerance induction. Deletion following administration of CTLA4Ig will be assessed using models in which antigen-responsive T cells may be monitored and quantified. If responding cells are deleted, we will investigate if CTLA4Ig induces apoptosis by altering expression of T cell survival genes such as bcl-2 and/or bcl-x. If deletion is incomplete or not detected, then we will attempt to reverse anergy with exogenous cytokines or Listeria infection and examine animals for the presence of veto cells. Suppression will be investigated using adoptive transfer studies, and as Th2 cytokine patterns are associated with tolerance, we will examine whether CTLA4Ig alters cytokine expression by responding T cells. These studies will allow Dr. Judge to gain experience in designing and executing a variety of studies essential for future immunologic research, and through critical evaluation of his work by Dr. Turka and other investigators here at Penn, he should attain a level of investigative excellence necessary for an independent research career.